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Introduction: Clear cell renal cell carcinoma (ccRCC) is characterized by a predominant metabolic reprogramming triggering energy production by anaerobic glycolysis at the expense of oxydative phosphorylation. Ketogenic diet (KD), which consists of high fat and low carbohydrate intake, could bring required energy substrates to healthy cells while depriving tumor cells of glucose. Our objective was to evaluate the effect of KD on renal cancer cell tumor metabolism and growth proliferation., Methods: Growth cell proliferation and mitochondrial metabolism of ACHN and Renca renal carcinoma cells were evaluated under ketone bodies (KB) exposure. In vivo studies were performed with mice (nude or Balb/c) receiving a xenograft of ACHN cells or Renca cells, respectively, and were then split into 2 feeding groups, fed either with standard diet or a 2:1 KD ad libitum. To test the effect of KD associated to immunotherapy, Balb/c mice were treated with anti-PDL1 mAb. Tumor growth was monitored., Results: In vitro , KB exposure was associated with a significant reduction of ACHN and Renca cell proliferation and viability, while increasing mitochondrial metabolism. In mice, KD was associated with tumor growth reduction and PDL-1 gene expression up-regulation. In Balb/c mice adjuvant KD was associated to a better response to anti-PDL-1 mAb treatment., Conclusion: KB reduced the renal tumor cell growth proliferation and improved mitochondrial respiration and biogenesis. KD also slowed down tumor growth of ACHN and Renca in vivo . We observed that PDL-1 was significantly overexpressed in tumor in mice under KD. Response to anti-PDL-1 mAb was improved in mice under KD. Further studies are needed to confirm the therapeutic benefit of adjuvant KD combined with immunotherapy in patients with kidney cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Richard, Beauvillain, Benoit, Barth, Aubert, Rolley, Bellal, Bourreau, Ferragu, Lebdai, Chevrollier, Henrion, Procaccio and Bigot.)

Dysregulated autophagy is associated with cardiovascular and metabolic diseases, where impaired flow-mediated endothelial cell responses promote cardiovascular risk. The mechanism by which the autophagy machinery regulates endothelial functions is complex. We applied multi-omics approaches and in vitro and in vivo functional assays to decipher the diverse roles of autophagy in endothelial cells. We demonstrate that autophagy regulates VEGF-dependent VEGFR signaling and VEGFR-mediated and flow-mediated eNOS activation. Endothelial ATG5 deficiency in vivo results in selective loss of flow-induced vasodilation in mesenteric arteries and kidneys and increased cerebral and renal vascular resistance in vivo. We found a crucial pathophysiological role for autophagy in endothelial cells in flow-mediated outward arterial remodeling, prevention of neointima formation following wire injury, and recovery after myocardial infarction. Together, these findings unravel a fundamental role of autophagy in endothelial function, linking cell proteostasis to mechanosensing., (© 2023. The Author(s).)

Tetrodotoxin (TTX) poisoning through the consumption of contaminated fish leads to lethal symptoms, including severe hypotension. This TTX-induced hypotension is likely due to the downfall of peripheral arterial resistance through direct or indirect effects on adrenergic signaling. TTX is a high-affinity blocker of voltage-gated Na + (Na V ) channels. In arteries, Na V channels are expressed in sympathetic nerve endings, both in the intima and media. In this present work, we aimed to decipher the role of Na V channels in vascular tone using TTX. We first characterized the expression of Na V channels in the aorta, a model of conduction arteries, and in mesenteric arteries (MA), a model of resistance arteries, in C57Bl/6J mice, by Western blot, immunochemistry, and absolute RT-qPCR. Our data showed that these channels are expressed in both endothelium and media of aorta and MA, in which scn2a and scn1b were the most abundant transcripts, suggesting that murine vascular Na V channels consist of Na V 1.2 channel subtype with Na V β1 auxiliary subunit. Using myography, we showed that TTX (1 µM) induced complete vasorelaxation in MA in the presence of veratridine and cocktails of antagonists (prazosin and atropine with or without suramin) that suppressed the effects of neurotransmitter release. In addition, TTX (1 µM) strongly potentiated the flow-mediated dilation response of isolated MA. Altogether, our data showed that TTX blocks Na V channels in resistance arteries and consecutively decreases vascular tone. This could explain the drop in total peripheral resistance observed during mammal tetrodotoxications.

Neonicotinoid insecticides are nicotine-derived molecules which exert acute neurotoxic effects over the insect central nervous system by activating nicotinic acetylcholine receptors (nAChRs). However, these receptors are also present in the mammalian central and peripheral nervous system, where the effects of neonicotinoids are faintly known. In mammals, cholinergic synapses are crucial for the control of vascular tone, blood pressure and skeletal muscle contraction. We therefore hypothesized that neonicotinoids could affect cholinergic networks in mammals and sought to highlight functional consequences of acute intoxication in rats with sub-lethal concentrations of the highly used acetamiprid (ACE) and clothianidin (CLO). In this view, we characterized their electrophysiological effects on rat α3β4 nAChRs, knowing that it is predominantly expressed in ganglia of the vegetative nervous system and the adrenal medulla, which initiates catecholamine secretion. Both molecules exhibited a weak agonist effect on α3β4 receptors. Accordingly, their influence on epinephrine secretion from rat adrenal glands was also weak at 100 μM, but it was stronger at 500 μM. Challenging ACE or CLO together with nicotine (NIC) ended up with paradoxical effects on secretion. In addition, we measured the rat arterial blood pressure (ABP) in vivo by arterial catheterization. As expected, NIC induced a significant increase in ABP. ACE and CLO did not affect the ABP in the same conditions. However, simultaneous exposure of rats to both NIC and ACE/CLO promoted an increase of ABP and induced a biphasic response. Modeling the interaction of ACE or CLO on α3β4 nAChR is consistent with a binding site located in the agonist pocket of the receptor. We present a transversal experimental approach of mammal intoxication with neonicotinoids at different scales, including in vitro, ex vivo, in vivo and in silico. It paves the way of the acute and chronic toxicity for this class of insecticides on mammalian organisms.

The importance of sexual dimorphism of the mouse brain metabolome was recently highlighted, in addition to a high regional specificity found between the frontal cortex, the cerebellum, and the brain stem. To address the origin of this dimorphism, we performed gonadectomy on both sexes, followed by a metabolomic study targeting 188 metabolites in the three brain regions. While sham controls, which underwent the same surgical procedure without gonadectomy, reproduced the regional sexual dimorphism of the metabolome previously identified, no sex difference was identifiable after gonadectomy, through both univariate and multivariate analyses. These experiments also made it possible to identify which sex was responsible for the dimorphism for 35 metabolites. The female sex contributed to the difference for more than 80% of them. Our results show that gonads are the main contributors to the brain sexual dimorphism previously observed, especially in females.

Background/objectives: Maternal obesity impacts vascular functions linked to metabolic disorders in offspring, leading to cardiovascular diseases during adulthood. Even if the relation between prenatal conditioning of cardiovascular diseases by maternal obesity and vascular function begins to be documented, little is known about resistance arteries. They are of particular interest because of their specific role in the regulation of local blood flow. Then our study aims to determine if maternal obesity can directly program fetal vascular dysfunction of resistance arteries, independently of metabolic disorders., Methods: With a model of rats exposed in utero to mild maternal diet-induced obesity (OMO), we investigated third-order mesenteric arteries of 4-month old rats in absence of metabolic disorders. The methylation profile of these vessels was determined by reduced representation bisulfite sequencing (RRBS). Vascular structure and reactivity were investigated using histomorphometry analysis and wire-myography. The metabolic function was evaluated by insulin and glucose tolerance tests, plasma lipid profile, and adipose tissue analysis., Results: At 4 months of age, small mesenteric arteries of OMO presented specific epigenetic modulations of matrix metalloproteinases (MMPs), collagens, and potassium channels genes in association with an outward remodeling and perturbations in the endothelium-dependent vasodilation pathways (greater contribution of EDHFs pathway in OMO males compared to control rats, and greater implication of PGI 2 in OMO females compared to control rats). These vascular modifications were detected in absence of metabolic disorders., Conclusions: Our study reports a specific methylation profile of resistance arteries associated with vascular remodeling and vasodilation balance perturbations in offspring exposed in utero to maternal obesity, in absence of metabolic dysfunctions.

Aim/hypothesis: In utero exposure to maternal diabetes increases the risk of developing hypertension and cardiovascular disorders during adulthood. We have previously shown that this is associated with changes in vascular tone in favor of a vasoconstrictor profile, which is involved in the development of hypertension. This excessive constrictor tone has also a strong impact on vascular structure. Our objective was to study the impact of in utero exposure to maternal diabetes on vascular structure and remodeling induced by chronic changes in hemodynamic parameters. Methods and Results: We used an animal model of rats exposed in utero to maternal hyperglycemia (DMO), which developed hypertension at 6 months of age. At a pre-hypertensive stage (3 months of age), we observed deep structural modifications of the vascular wall without any hemodynamic perturbations. Indeed, in basal conditions, resistance arteries of DMO rats are smaller than those of control mother offspring (CMO) rats; in addition, large arteries like thoracic aorta of DMO rats have an increase of smooth muscle cell attachments to elastic lamellae. In an isolated perfused kidney, we also observed a leftward shift of the flow/pressure relationship, suggesting a rise in renal peripheral vascular resistance in DMO compared to CMO rats. In this context, we studied vascular remodeling in response to reduced blood flow by in vivo mesenteric arteries ligation. In DMO rats, inward remodeling induced by a chronic reduction in blood flow (1 or 3 weeks after ligation) did not occur by contrast to CMO rats in which arterial diameter decreased from 428 ± 17 μm to 331 ± 20 μm (at 125 mmHg, p = 0.001). In these animals, the transglutaminase 2 (TG2) pathway, essential for inward remodeling development in case of flow perturbations, was not activated in low-flow (LF) mesenteric arteries. Finally, in old hypertensive DMO rats (18 months of age), we were not able to detect a pressure-induced remodeling in thoracic aorta. Conclusions: Our results demonstrate for the first time that in utero exposure to maternal diabetes induces deep changes in the vascular structure. Indeed, the early narrowing of the microvasculature and the structural modifications of conductance arteries could be a pre-emptive adaptation to fetal programming of hypertension.

Diabetes Mellitus is associated with severe cardiovascular disorders involving the renin-angiotensin system, mainly through activation of the angiotensin II type 1 receptor (AT1R). Although the type 2 receptor (AT2R) opposes the effects of AT1R, with vasodilator and anti-trophic properties, its role in diabetes is debatable. Thus we investigated AT2R-mediated dilatation in a model of type 1 diabetes induced by streptozotocin in 5-month-old male mice lacking AT2R (AT2R -/y ). Glucose tolerance was reduced and markers of inflammation and oxidative stress (cyclooxygenase-2, gp91phox p22phox and p67phox) were increased in AT2R -/y mice compared to wild-type (WT) animals. Streptozotocin-induced hyperglycaemia was higher in AT2R -/y than in WT mice. Arterial gp91phox and MnSOD expression levels in addition to blood 8-isoprostane and creatinine were further increased in diabetic AT2R -/y mice compared to diabetic WT mice. AT2R-dependent dilatation in both isolated mesenteric resistance arteries and perfused kidneys was greater in diabetic mice than in non-diabetic animals. Thus, in type 1 diabetes, AT2R may reduce glycaemia and display anti-oxidant and/or anti-inflammatory properties in association with greater vasodilatation in mesenteric arteries and in the renal vasculature, a major target of diabetes. Therefore AT2R might represent a new therapeutic target in diabetes.

Mutations in the filamin A (FlnA) gene are frequently associated with severe arterial abnormalities, although the physiological role for this cytoskeletal element remains poorly understood in vascular cells. We used a conditional mouse model to selectively delete FlnA in smooth muscle (sm) cells at the adult stage, thus avoiding the developmental effects of the knockout. Basal blood pressure was significantly reduced in conscious smFlnA knockout mice. Remarkably, pressure-dependent tone of the resistance caudal artery was lost, whereas reactivity to vasoconstrictors was preserved. Impairment of the myogenic behavior was correlated with a lack of calcium influx in arterial myocytes upon an increase in intraluminal pressure. Notably, the stretch activation of CaV1.2 was blunted in the absence of smFlnA. In conclusion, FlnA is a critical upstream element of the signaling cascade underlying the myogenic tone. These findings allow a better understanding of the molecular basis of arterial autoregulation and associated disease states., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Epidemiologic studies have demonstrated that cardiovascular risk is not only determined by conventional risk factors in adulthood, but also by early life events which may reprogram vascular function. To evaluate the effect of maternal diabetes on fetal programming of vascular tone in offspring and its evolution during adulthood, we investigated vascular reactivity of third order mesenteric arteries from diabetic mother offspring (DMO) and control mother offspring (CMO) aged 3 and 18 months. In arteries isolated from DMO the relaxation induced by prostacyclin analogues was reduced in both 3- and 18-month old animals although endothelium (acetylcholine)-mediated relaxation was reduced in 18-month old DMO only. Endothelium-independent (sodium nitroprusside) relaxation was not affected. Pressure-induced myogenic tone, which controls local blood flow, was reduced in 18-month old CMO compared to 3-month old CMO. Interestingly, myogenic tone was maintained at a high level in 18-month old DMO even though agonist-induced vasoconstriction was not altered. These perturbations, in 18-months old DMO rats, were associated with an increased pMLC/MLC, pPKA/PKA ratio and an activated RhoA protein. Thus, we highlighted perturbations in the reactivity of resistance mesenteric arteries in DMO, at as early as 3 months of age, followed by the maintenance of high myogenic tone in older rats. These modifications are in favour of excessive vasoconstrictor tone. These results evidenced a fetal programming of vascular functions of resistance arteries in adult rats exposed in utero to maternal diabetes, which could explain a re-setting of vascular functions and, at least in part, the occurrence of hypertension later in life.

Many factors, including hyperglycemia, hypertension, obesity, dyslipidemia, and a sedentary lifestyle, contribute to a high prevalence of cardiovascular disease. Specific vascular impairment treatments in the context of diabetes and vascular risk need to be improved. Salidroside is the primary active component of Rhodiola rosea and has documented antioxidative, cardioprotective, and vasculoprotective properties. The aim of this study was to test the hypothesis that salidroside has protective effects against hyperglycemia, hypertension, and vasodilation impairment in the Goto-Kakizaki (GK) rat model of diabetes. We evaluated cardiovascular parameters (e.g., daytime/nighttime systolic and diastolic blood pressure, heart rate, and activity), metabolic parameters (e.g., body weight, food and water consumption, serum fructosamine level, glucose tolerance), eNOS / phospho-eNOS expression level and in vitro vascular reactivity of aorta and second-order mesenteric arteries in Wistar-Kyoto (control) and GK (diabetic) rats treated with salidroside (40 mg/kg) or placebo (water) for 5 weeks. GK rats showed hypertension, marked glucose intolerance, and impaired endothelium-dependent and endothelium-independent vasodilation capacity. Salidroside showed beneficial effects on endothelial and non-endothelial vasodilation and likely acts on the endothelium and smooth muscle cells through the soluble guanylyl cyclase pathway. Despite its vascular effects, salidroside had no effect on blood pressure and heart rate in GK and control rats, it did not improve glucose metabolism or limit hypertension in the GK model of type 2 diabetes.

The most accepted animal model for simulation of the physiological and morphological consequences of microgravity on the cardiovascular system is one of head-down hindlimb unloading. Experimental conditions surrounding this model include not only head-down tilting of rats, but also social and restraint stresses that have their own influences on cardiovascular system function. Here, we studied levels of spontaneous locomotor activity, blood pressure, and heart rate during 14 days under the following experimental conditions: cage control, social isolation in standard rat housing, social isolation in special cages for hindlimb unloading, horizontal attachment (restraint), and head-down hindlimb unloading. General activity and hemodynamic parameters were continuously monitored in conscious rats by telemetry. Heart rate and blood pressure were both evaluated during treadmill running to reveal cardiovascular deconditioning development as a result of unloading. The main findings of our work are that: social isolation and restraint induced persistent physical inactivity, while unloading in rats resulted in initial inactivity followed by normalization and increased locomotion after one week. Moreover, 14 days of hindlimb unloading showed significant elevation of blood pressure and slight elevation of heart rate. Hemodynamic changes in isolated and restrained rats largely reproduced the trends observed during unloading. Finally, we detected no augmentation of tachycardia during moderate exercise in rats after 14 days of unloading. Thus, we concluded that both social isolation and restraint, as an integral part of the model conditions, contribute essentially to cardiovascular reactions during head-down hindlimb unloading, compared to the little changes in the hydrostatic gradient.

The aim of the present study was to investigate the role of mitochondrial nitric oxide synthase (mtNOS) in the septic shock and analyze its relationship to ventricular contractility. Two models of septic shock [lipopolysaccharide (LPS)-induced and cecal ligation and puncture (CLP)-induced] were used. There was a significant depression of ventricular contractile parameters recorded in the late stage of the septic shock. After measurement of ventricular-dynamic parameters, mitochondrial and cytoplasmic fractions were isolated and their nitric oxide synthase (NOS) activity was assessed using a NOS activity assay kit. Both models showed a larger increase in mitochondrial NOS activity than that in cytosol. However, the increase in mtNOS activity in the LPS-induced shock model was less pronounced than in the CLP-induced model. Regression analysis shows that mitochondrial nitric oxide synthase (mtNOS) activity is negatively correlative to the left ventricular developed pressure in CLP model. The results suggest that mitochondrial NOS may mainly contribute to the ventricular depression in the septic shock.

Full muscarinic stimulation in bovine tracheal smooth muscle caused a sustained contraction and increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) that was largely resistant to inhibition by nifedipine. Depletion of internal Ca(2+) stores with cyclopiazonic acid resulted in an increased efficacy of nifedipine to inhibit this contraction and the associated increase in [Ca(2+)](i). Thus internal Ca(2+) store depletion promoted electromechanical coupling between full muscarinic stimulation and muscle contraction to the detriment of pharmacomechanical coupling. A similar change in coupling mode was induced by ryanodine even when it did not significantly modify the initial transient increase in [Ca(2+)](i) induced by this stimulation, indicating that depletion of internal stores was not necessary to induce the change in excitation-contraction coupling mode. Blockade of the Ca(2+)-activated K(+) channel by tetraethylammonium, charybdotoxin, and iberiotoxin all induced the change in excitation-contraction coupling mode. These results suggest that in this preparation, Ca(2+) released from the ryanodine-sensitive Ca(2+) store, by activating Ca(2+)-activated K(+) channels, plays a central role in determining the expression of the pharmacomechanical coupling mode between muscarinic excitation and the Ca(2+) influx necessary for the maintenance of tone.

Aim: To assess the cytotoxic effects of cobra cardiotoxin (CTX) on rat aorta., Methods: Measure of contractility of aortic rings with or without endothelium., Results: In endothelium-intact rings, CTX 10 mumol.L-1 induced a transient relaxation followed by a sustained contraction. Removal of the endothelium or pre-incubation of the rings with NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) abolished the transient relaxation but did not affect the magnitude of the contractile response induced by CTX. CTX itself induced contraction of vascular smooth muscle but also reduced contractions induced by phenylephrine (PhE) or KCl stimulation in a concentration-dependent manner. Contraction induced by CTX was dependent on the external Ca2+ concentration. Maximal contractile response to CTX was obtained in medium containing Ca2+ 1 mmol.L-1. This response decreased with higher Ca2+ concentration and disappeared when Ca2+ 7 mmol.L-1, organic and inorganic calcium channel blockers were present in the external solution before CTX addition. In preparations with the endothelium intact and incubated with CTX, relaxation by acetylcholine (ACh) stimulation of the tension induced by PhE was decreased. Endothelium-dependent relaxation to ACh was preserved when Ca2+ 5 mmol.L-1 was added to the medium prior to CTX., Conclusion: CTX first triggers the release of NO from the endothelium which results in muscle relaxation, and then causes smooth muscle contraction, Ca2+ and Ca2+ channel blockers prevented the effect of CTX.